Long noncoding RNA ADAMTS9-AS1 represses ferroptosis of endometrial stromal cells by regulating the miR-6516-5p/GPX4 axis in endometriosis

Endometriosis (EMs) is one of the most frequent diseases of reproductive-age women and is characterized by the growth of endometrial tissues beyond the uterus. The enhanced proliferative and migratory potential of endometrial stromal cells (ESCs) plays an important role in the progression of EMs. Mounting studies have demonstrated that long noncoding RNAs (lncRNAs) exert an important role in regulating the development and progression of EMs. Given the aberrant expression of lncRNA ADAMTS9-AS1 in ectopic endometrium (ecEM), we investigated the biological effect of ADAMTS9-AS1 on ESC proliferation and migration and explored the underlying mechanism. The current data showed that ADAMTS9-AS1 expression was significantly upregulated in ecEM compared with eutopic endometrium (euEM) in patients with EMs and in a murine model of EMs. Functionally, ADAMTS9-AS1 knockdown in ectopic ESCs (EESCs) decreased cell viability and migration, whereas ADAMTS9-AS1 overexpression in normal ESCs (NESCs) enhanced cell viability and migration. More importantly, the effect of ADAMTS9-AS1 inhibition on decreasing ESC viability was significantly blocked by ferrostatin-1 (Fer-1, a ferroptosis inhibitor), and ADAMTS9-AS1 overexpression repressed erastin (a ferroptosis activator)-induced cell death. Furthermore, the regulatory role of ADAMTS9-AS1 in ferroptosis was defined and evidenced by increased reactive oxygen species (ROS) levels and malonyl dialdehyde (MDA) content and decreased expression of glutathione peroxidase 4 (GPX4) after ADAMTS9-AS1 inhibition. Mechanistically, ADAMTS9-AS1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-6516-5p to derepress the expression of GPX4, the critical repressor of ferroptosis. Taken together, these results demonstrate that upregulated ADAMTS9-AS1 accelerates ESC proliferation and migration by regulating miR-6516-5p/GPX4-dependent ferroptosis and may be a potential target for the treatment of EMs.

expression is closely correlated with a variety of biological processes and human diseases, such as chromosome dose compensation, development, tumorigenesis, and cardiovascular diseases [8][9][10][11] . For example, Xu et al. reported that 172 lncRNAs and 188 mRNAs are differentially expressed in endometrial carcinoma tissues compared with normal tissues 12 . Several lncRNAs (KIAA0087, RP11-501O2, FAM212B-AS1, etc.) might be a key regulator of endometrial carcinogenesis and progression. Emerging evidence has also revealed the role of lncRNAs in the pathogenesis of EMs. A total of 576 differentially expressed lncRNAs were identified in the ectopic endometrium (ecEM) of adenomyosis, of which 388 lncRNAs were increased and 188 lncRNAs were decreased 13 . Zhang et al. demonstrated that the expression of lncRNA CCDC144NL-AS1 is upregulated in ectopic endometrial tissues compared with paired eutopic endometrial and normal endometrial (NE) tissues 14 . Functional studies further showed that CCDC144NL-AS1 inhibition decreases endometrial stromal cell (ESC) migration and invasion 14 .
The role of lncRNA ADAMTS9-AS1 (hereafter named ADAMTS9-AS1) in tumor progression has been widely investigated. Low expression of ADAMTS9-AS1 predicts poorer survival than higher ADAMTS9-AS1 expression in patients with prostate cancer, and ADAMTS9-AS1 inhibits prostate cancer cell proliferation, indicating that ADAMTS9-AS1 functions as a tumor suppressor in prostate cancer 15 . A recent study showed that ADAMTS9-AS1 is significantly increased in ectopic endometrial tissues 16 . However, the biological role of ADAMTS9-AS1 in EMs remains unknown.
Ferroptosis is an iron-dependent nonapoptotic type of cell death that distinctly differs from other forms of cell death, including apoptosis, pyroptosis, senescence and autophagy [17][18][19][20] . Overproduction of iron-induced lipid reactive oxygen species (ROS) is the crucial event in ferroptosis 21,22 . Although the levels of iron and lipid peroxide content are increased in the peritoneal fluid of women with EMs compared to those without [23][24][25] , the role of ferroptosis in EMs has not been systematically investigated. In this study, we demonstrated that ADAMTS9-AS1 was significantly upregulated in ecEM compared with eutopic endometrium (euEM). ADAMTS9-AS1 knockdown decreased cell viability and migration, whereas the effect was blocked by a ferroptosis inhibitor. ADAMTS9-AS1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-6516-5p to derepress the expression of GPX4.

Materials and methods
A murine model of EMs. Animal experiments were approved by the Experimental Animal Committee of Shanghai Municipal Hospital of Traditional Chinese Medicine and carried out according to Shanghai Municipal Hospital of Traditional Chinese Medicine guidelines for the use of animals. All animal experiments were performed in accordance with the ARRIVE guidelines as described previously 26 to minimize the number of animals used in the study and animal suffering. Female BALB/c mice (4-6 weeks old, 18-20 g) were obtained from Shanghai Regan Biotechnology Co., Ltd. (Shanghai, China) and were reared in a specific, pathogen-free facility [27][28][29] . After 1 week of acclimatization, mice were randomly divided into two groups: the donor group (n = 10) and recipient groups (n = 10). Ovariosteresis and estradiol valerate injection (0.5 µg/mouse/week; Aladdin, Shanghai, China) was carried out to avoid differences in the estrous cycle 28 . Mice were anesthetized by 2% isoflurane, and then the ovaries on both sides were exposed through flank incisions and removed. Donor mice were sacrificed under isoflurane anesthesia, and each uterine horn of the donor mice was concentrated and peeled in warm PBS to remove uterine muscle. Endometrial tissues were weighed and cut into small fragments with scissors and resuspended in sterile PBS with 1 × ampicillin (Beyotime, Shanghai, China). After that, endometrium preparation was intraperitoneally injected into two recipient mice (50 mg/mouse). Two weeks after EM transplantation, endometriosis lesions and eutopic endometrial tissues were removed from the peritoneal cavities and uteri.
Clinical samples. Human ectopic and eutopic endometrial tissues (n = 17) were provided by Shanghai Municipal Hospital of Traditional Chinese Medicine with written informed consent signed by the patients according to the Declaration of Helsinki Principles. All of the protocols in this study were approved by Shanghai Municipal Hospital of Traditional Chinese Medicine. Primary ESCs. Primary murine ESCs were isolated from the eutopic and ectopic endometrial tissues.
Total RNA extraction and quantitative RT-PCR (qPCR) assay. Total RNA was extracted from endometrial tissues or ESCs with RNAiso Plus (TaKaRa, Shiga, Japan) and quantitated by Multiskan SkyHigh (Thermo Fisher Scientific). A Prime Script RT-PCR Kit (TaKaRa) was applied to carry out reverse transcription PCR (RT-PCR). The RT-PCR protocol was 65 °C for 5 min, 30 °C for 10 min, and 42 °C for 30 min. qPCR was performed using Accurate 96 (DLAB, China) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The program setting was based on the manufacturer's recommendations, and the thermocycling conditions were as follows: initial denaturation at 95 °C for 10 s, denaturation at 95 °C for 10 s, and annealing and extension at 65 °C for 10 s; the reaction was performed for 30 cycles. Each reaction was carried out at least in triplicate. ADAMTS9-AS1 and GPX4 RNA transcription was quantified by the 2 −ΔΔCt method and normalized to endogenous β-actin mRNA. The primer sequences are listed in Supporting Table S1. www.nature.com/scientificreports/ Overexpression and RNA interference (RNAi). The recombinant construct (pcDNA-ADAMTS9-AS1) containing the full-length ADAMTS9-AS1 sequence was constructed by inserting the open reading frame into the pcDNA vector. miR-6516-5p mimics were used to mimic the overexpression of miR-6516-5p. siRNA-ADAMTS9-AS1 (siADAMTS9-AS1) was used to knockdown ADAMTS9-AS1 in ESCs, and siRNA-control (N.C) was used as a control. The ESCs were seeded into culture dishes before transfection until the cells adhered on the second day. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was applied to prepare a mixture of siADAMTS9-AS1, N.C, miR-6516-5p or pcDNA-ADAMTS9-AS1 with Lipofectamine 3000 according to the instructions.

Transwell migration assay.
To explore the effect of ADAMTS9 AS1 and miR-6516-5p on regulating ESC migration, 24-well Transwell chambers (Corning, NY, USA) with 8-μm pores were used to assess the migration of EESCs. In brief, the transfected EESCs and NESCs were seeded into the upper chamber at a density of 200 μL 2.5 × 10 4 cells with DMEM/12F medium without serum, and 700 μL of DMEM/12F medium supplemented with 10% FBS was added into the lower chamber. After incubating for 48 h at 37 °C with 5% CO 2 , the cells on the lower surface of the membranes were fixed with 4% paraformaldehyde (Beyotime) and stained with 0.1% crystal violet (Beyotime) for 10 min. Images were obtained using BZ-X700 (Keyenth, Osaka, Japan), and the number of cells migrating to independent areas was counted using Fiji software (ImageJ 1.51s, NIH, USA).
TUNEL staining. Cell death was assessed using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). In brief, after being transfected with si-ADAMTS9-AS1 or siRNA-control for 48 h, EESCs were fixed in 4% formaldehyde and then stained using the TUNEL mixture (Elabscience, Wuhan, China) for 60 min at 37 °C. Cell nuclei was counterstained with DAPI (Beyotime) for 5 min. Cells were observed using a fluorescence microscopy (DMI4000B, Leica).
Measure of iron concentration. Intracellular iron concentrations were measured using an iron assay kit (ab83366, Abcam) according to the manufacturer's instructions. Briefly, ESCs were lysed with iron assay buffer (3 × 10 3 cells/well) in a final volume of 100 μL, treated with 5 μL of iron reducer, plated in 96-well plates and incubated at room temperature for 30 min. Then, 100 μL of iron probe was added to mark ferrous ions. The absorbance was measured at 593 nm with a Multiskan SkyHigh (Thermo Fisher Scientific).
GSH and MDA content. The malondialdehyde (MDA) content of the ESCs was assayed using a lipid peroxidation assay kit (ab118970, Abcam, MA, USA) after ADAMTS9-AS1 knockdown or overexpression in the presence or absence of miR-6516-5p or siGpx4. The glutathione (GSH) content in the ESCs was assayed using a glutathione assay kit (ab65322, Abcam).

Lipid ROS level.
Lipid ROS levels were tested using a fluorescent-labeled oxidation-sensitive probe (C11-BODIPY, Thermo Fisher Scientific) as previously described 31 . In brief, ESCs (5 × 10 5 ) were plated in 24-well plates and treated with the indicated reagents. RNA-pulldown assay. Biotin-labeled miR-6516-5p and miR-cont were transcribed in vitro using the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific). Approximately 2 × 10 7 cells were dissolved in standard lysis buffer (Thermo Fisher Scientific) plus 10 U/mL RNase inhibitor (Beyotime). Next, streptavidinlabeled beads were added to each binding reaction and incubated for 90 min. Finally, ADAMTS9 AS1 in the eluate was quantified by qPCR.
Statistical analysis. The data were analyzed using GraphPad Prism v.7.0 (CA, USA) and presented as the mean ± SEM from at least three independent experiments. The significance between the different groups was assessed using Student's t test or one-way ANOVA followed by a Tukey-Kramer multiple comparisons test. P values less than 0.05 were considered to indicate a statistically significant difference.

ADAMTS9-AS1 expression was significantly upregulated in ecEM.
The expression level of ADAMTS9-AS1 is dysregulated in ecEM 16 and in several types of cancer 15,32,33 . To investigate the biological function of ADAMTS9-AS1 in EMs, the expression pattern of ADAMTS9-AS1 was verified in endometrial tissues. The data from qPCR analysis in seventeen paired ecEM and euEM tissues showed that the expression level of ADAMTS9-AS1 was significantly increased in ecEM compared to euEM (Fig. 1A). To further validate the results, a murine model of EMs was established, and ecEM and euEM tissues were collected. Figure 1B showed that ADAMTS9-AS1 expression was also upregulated in ecEM compared to euEM in a murine model of EMs. Then, primary EESCs and NESCs were collected from the ecEM and euEM tissues, respectively, and the ADAMTS9-AS1 level was assessed. As shown in Fig. 1C, the ADAMTS9-AS1 level in the EESCs was higher than that in the NESCs.

ADAMTS9-AS1 accelerated ESC growth and migration.
To explore the effect of ADAMTS9-AS1 on regulating ESC viability and migration, ADAMTS9-AS1 was knocked down in EESCs and overexpressed in NESCs (Supporting Fig. S1A,B). Figure 2A showed that ADAMTS9-AS1 knockdown in EESCs significantly decreased cell viability. The results from Tunel assay revealed that ADAMTS9-AS1 knockdown resulted in obvious cell death (Fig. 2B). In addition, the migratory behavior of ESCs plays a crucial role in EMs pathogenesis 34 . Figure 2C,D showed that ADAMTS9-AS1 knockdown in EESCs significantly repressed cell migration. In contrast, ADAMTS9-AS1 overexpression in NESCs increased cell viability (Fig. 2E) and facilitated cell migration (Fig. 2F,G). These data suggest that upregulated ADAMTS9-AS1 increases ESC viability and promotes ESC migration.

ADAMTS9-AS1 repressed ferroptosis of ESCs.
To investigate the pattern of ADAMTS9-AS1-induced ESC death, EESCs were treated with ADAMTS9-AS1-specific siRNA (siADAMTS9-AS1) in the presence of Fer-1 (a specific inhibitor of ferroptosis), ZVAD-FMK (a specific inhibitor of apoptosis), 3-MA (a specific inhibitor of autophagy), or disulfiram (a specific inhibitor of pyroptosis). Forty-eight hours later, cell viability was assessed using CCK-8. Figure 3A showed that the siADAMTS9-AS1-mediated decrease in EESC viability was significantly prevented by Fer-1 and ZVAD-FMK but not necrostatin-1, 3-MA or disulfiram, suggesting  www.nature.com/scientificreports/ that ADAMTS9-AS1 knockdown accelerated EESC death by triggering apoptosis and ferroptosis. The role of ADAMTS9-AS1 in regulating ferroptosis of EESCs was next explored because the effect of Fer-1 on regulating EESC viability was more significant than that of ZVAD-FMK. Figure 3B further revealed that erastin (a specific activator of ferroptosis) treatment decreased NESC viability, whereas ADAMTS9-AS1 overexpression blocked this effect. Fer-1 also prevented sorafenib-induced EESC death (Fig. 3C). These data demonstrate that ferroptosis is an important form of ESC death and that ADAMTS9-AS1 is negatively correlated with ESC ferroptosis. We then compared the ferroptosis level between NESCs and EESCs. Figure 4A-C showed that the relative iron concentration, MDA content, and ROS level were lower in EESCs than in NESCs, indicating that ferroptosis was repressed in EESCs. To determine whether ADAMTS9-AS1 knockdown triggered ferroptosis of EESCs, ROS levels and MDA content were assessed after ADAMTS9-AS1 knockdown. Figure 4D,E showed that ADAMTS9-AS1 inhibition resulted in a significant increase in ROS and MDA levels, suggesting that ADAMTS9-AS1 negatively regulated ferroptosis of EESCs. To uncover the mechanism by which ADAMTS9-AS1 repressed ferroptosis, the expression level of ferroptosis-related genes (COX-2, ACSL4, PTGS2, NOX1, GPX4, and FTH1) were assayed in EESCs after ADAMTS9-AS1 depletion. Figure 4F-H showed that ADAMTS9-AS1 depletion significantly decreased the mRNA and protein expression levels of GPX4, a key repressor of ferroptosis. These data indicate that ADAMTS9-AS1 negatively regulates ferroptosis of ESCs, possibly by regulating GPX4. ADAMTS9-AS1 acted as a ceRNA by sponging miR-6516-5p. LncRNAs located in the cytoplasm commonly function as ceRNAs to indirectly control mRNA expression by absorbing miRNAs 35,36 . To explore the mechanism underlying ADAMTS9-AS1 in regulating ferroptosis, the subcellular localization of ADAMTS9-AS1 in ESCs was first assessed using FISH. As shown in Fig. 5A, ADAMTS9-AS1 was primarily located in the cyto- , and ROS levels (C) were assayed using the indicated kits for NESCs and EESCs. The ROS levels (D) and MDA content (E) were assessed using the indicated kits for EESCs after ADAMTS9-AS1 knockdown. (F) The mRNA levels of COX-2, ACSL4, PTGS2, NOX1, GPX4, and FTH1 were assayed using qPCR analysis in EESCs after ADAMTS9-AS1 knockdown. (G,H) The protein level of GPX4 was assayed using western blot analysis in EESCs after ADAMTS9-AS1 knockdown. *p < 0.05, **p < 0.01.  37 was used to predict the potential miRNAs absorbed by ADAMTS9-AS1. A total of 104 miRNAs were predicted through the miRDB tool (Supporting Table S2). Based on the above results that ADAMTS9-AS1 positively regulated GPX4 expression, miRNAs targeting GPX4 were screened using TargetScan 7.2 (http:// www. targe tscan. org/ vert_ 72/). miR-6516-5p exhibited the potential to be involved in ADAMTS9-AS1/miR-6516-5p/GPX4 ceRNA networks (Fig. 5B,F).

Discussion
Iron (Fe) is an indispensable element that regulates cell survival and proliferation, and it has been reported that Fe shortages are correlated with many reproductive diseases 20 . Paradoxically, Fe overload exerts multiple effects on EMs progression, including oxidative stress or lesion proliferation 38 . As an Fe-dependent nonapoptotic form www.nature.com/scientificreports/ of cell death, the biological role of ferroptosis in EMs is gradually being revealed. Ferroptosis resistance is correlated with ESC growth and proliferation, and ferroptosis-associated genes may possess clinical potential in the diagnosis and treatment of EMs. In the present study, we demonstrate that (i) ADAMTS9-AS1 expression was significantly increased in the ecEM, (ii) ADAMTS9-AS1 accelerated ESC growth and migration, (iii) ADAMTS9-AS1 repressed ferroptosis of ESCs, (iv) ADAMTS9-AS1 acted as a ceRNA by sponging miR-6516-5p, and (v) ADAMTS9-AS1 increased GPX4 expression in a miR-6516-5p-dependent manner. These results demonstrated the important function of the ADAMTS9-AS1/miR-6516-5p/GPX4/ferroptosis axis in regulating ESC viability and migration and may provide a potential opportunity for EMs therapy. The role of ADAMTS9-AS1 in tumorigenesis and progression has been widely investigated. Through lncRNA expression profiling analysis, Wang et al. first demonstrated that the ADAMTS9-AS1 level is significantly increased in epithelial ovarian cancer tissues compared with normal tissues 39 . ADAMTS9-AS1 overexpression represses prostate cancer cell proliferation, and low expression levels of ADAMTS9-AS1 predict poorer survival than higher ADAMTS9-AS1 expression levels, suggesting that ADAMTS9-AS1 functions as a tumor suppressor in prostate cancer 15 . In contrast, ADAMTS9-AS1 facilitates cancer cell proliferation and migration in hepatocellular carcinoma and colorectal cancer 40,41 . These studies indicate that ADAMTS9-AS1 might exert different biological effects in different types of cells. In particular, a recent study showed that ADAMTS9-AS1 expression is dysregulated in ectopic endometrial tissues 16 . Based on these facts, we investigated the biological role of ADAMTS9-AS1 in the pathogenesis of EMs. The current results revealed that ADAMTS9-AS1 expression is significantly increased in ecEM compared with euEM in patients with EMs and in a murine model of EMs. Functional studies demonstrated that ADAMTS9-AS1 inhibition in ESCs represses cell viability and migration, whereas ADAMTS9-AS1 overexpression increases cell viability and migration, indicating that ADAMTS9-AS1 functions as an activator of EMs.
The underlying mechanism by which ADAMTS9-AS1 facilitates ESC viability was further investigated. As an antisense lncRNA, ADAMTS9-AS1 cannot affect the expression of overlapping coding genes (ADAMTS9, data not shown) 39 , indicating that ADAMTS9-AS1 possesses other modes of function in EMs. Emerging studies have demonstrated that lncRNAs can interact with miRNAs as ceRNAs to regulate target mRNAs 42 . The ceRNA hypothesis has also been confirmed in EMs. Wang et al. identified an EMs-related ceRNA network involving forty-five pathways and several ceRNAs as potential biomarkers for endometrial receptivity 43 . LINC01541 sponges miR-506-5p to regulate its downstream Wnt/β-Catenin pathway in EMs and thus inhibits the proliferation and invasion of ESCs 44 . In this study, we demonstrated that ADAMTS9-AS1 functions as a ceRNA by sponging miR-6516-5p to derepress the expression of GPX4.
It is well known that GPX4 is the critical repressor of ferroptosis 45 . As a glutathione-dependent enzyme, GPX4 converts toxic lipid hydroperoxides to nontoxic lipid alcohols 46 and thus decreases Fe-induced conversion of lipid hydroperoxides to highly reactive lipid alkoxy radicals. The genetic variants of GPX4 are different between women with advanced stage EMs and mild EMs 47 . For example, GPX4 rs713041 is associated with the severity of EMs, indicating that abnormal GPX4 is involved in the pathogenesis of EMs. Moreover, the regulatory role of lncRNAs in GPX4 expression has been verified. Bai et al. demonstrated that H19 knockdown decreases GPX4 expression and facilitates ferroptosis in spontaneous abortion 48 . Based on these analyses, we investigated whether ADAMTS9-AS1 regulates ferroptosis resistance in EMs through the miRNA/GPX4 axis. We demonstrated that ADAMTS9-AS1 accelerates ESC proliferation and migration by regulating miR-6516-5p/ GPX4-dependent ferroptosis.

Data availability
All data generated or analyzed during this study are included in this published article (and its Supplementary  Information files).